Pierce’s Disease was reported for the first time in Costa Rica in 1979; the causal agent was isolated in 1980. The pathogen was known as Pierce’s Disease bacteria and was identified as Xylella fastidiosa. The presence of X. fastidiosa was confirmed in grapevine in three zones of Costa Rica. In each zone leaves with similar symptoms to Pierce Disease were collected. The samples were positive by DAS-ELISA using specific antibodies for X. fastidiosa (Agdia Inc. Elkhart Indiana). Rod-shaped, Gram negative, catalase positive, oxidase negative bacteria were isolated from petioles using PW medium. Bacterial colonies were visible 6 to 7 days. Colonies were smooth, opalescent, with entire margins and a diameter of 2 to 3 mm. DNA was extracted from purified isolates and PCR was performed using 272-1-int, 272-2-int and RST31, RST33. Bands of 500bp and a 733pb were amplified respectively. Electron micrographs showed bacteria 1–4 µm long with a cell wall with ridges and furrows identical to the morphology reported for X. fastidiosa.

Onion (Allium cepa L.) is fourth in economic importance among horticultural crops in Puerto Rico. Bulbs and roots are attacked by various pathogens and affect production. The objective was to characterize eighteen Phoma soil isolates based on morphology, pathogenicity, and molecular characteristics. Isolates were on potato dextrose, oatmeal, and malt agar. Semi-permanent slides were done to observe hyphae, conidia, pycnidia, chlamydospores, and swollen cells. Pathogenicity tests were run in the laboratory and field using ‘Mercedes’ and ‘Excalibur’. Observed symptomatic plants were used for isolations. Phoma putaminum Speg. and possibly P. macrostoma var. incolorata were identified and are first reports in Puerto Rico and the Caribbean. Pathogenicity tests in vitro showed severe necrosis in young bulbs and roots with P. putaminum and pink root rot with P. macrostoma var. incolorata. Field tests are in progress. Genomic DNA was extracted to amplify the ITS region of the rDNA using PCR. Restriction enzyme Hind III created a polymorphic pattern from the amplified rDNA separating both species. Sequencing the rDNA and phylogenic analysis is planned.

Little is known on the importance of bacterial diseases occurring in onions in Puerto Rico. Foliage from several fields was sampled during a full growing season. Fifty foliar isolates were identified using the Biolog® method, 19 of which may have phytopathogenic potential. We determined that six of eight isolates were pathogenic, as verified using field and laboratory tests. The pathogenic bacteria were the following genera: Pseudomonas, Xanthomonas, Acidovorax, Burkholderia, and Pantoea.

Onion is an important crop in Puerto Rico, where its production is limited by pathogenic fungi. Sampling from commercial fields yielded isolates of Fusarium (140), Alternaria (18), and Phoma (18).Among these, the most prevalent were: Fusarium solani and F. equiseti; Alternaria tennuisima and several isolates of Alternaria “semiarborescent” group; Phoma putaminun and Phoma macrostoma var. incolorata. For molecular characterization of these isolates, we amplified rDNA’s ITS region and performed restriction fragment length polymorphisms (RFLP) analyses. Patterns of restriction were converted into a binary matrix to construct dendograms under multivariate analysis using Jaccard’s distance. Phylogenetic trees based on ITS sequences were used for comparisons among Fusarium isolates.

The interaction of endophytic bacteria with citrus and citrus variegated chlorosis (CVC) was previously analyzed. X. fastidiosa is the cause of crespera of coffee and is associated with CVC-like symptoms in avocado. Bacteria were obtained during attempts to isolate X. fastidiosa from symptomatic coffee and avocado. Coffee strains and some avocado strains were characterized by colony morphology, Gram stain, biochemical assays, and electron microscopy. M. mesophilicum was isolated from symptomatic avocados and 10 trees were PCR positive. Pseudomonas syringae-glycinea, Pantoea agglomerans, C. flaccumfaciens-betae/oortii, Moraxella nonliquefaciens, and Enterobacter intermedius were isolated and identified by fatty-acid methyl ester analysis. Plans are to confirm them as endophytes, determine their identity/frequency, and determine an interaction with X. fastidiosa in crespera in the two diseases.

Incidence and morphological characterization of Alternaria spp. occurring in onions in southern Puerto Rico. J. FERNANDEZ, L. I. Rivera, and I. Cabrera. P.O. Box 9030, Department of Crop Protection, University of Puerto Rico-Mayaguez Campus, Mayaguez, P.R. 00681.Alternaria late blight of onions, causes losses in Puerto Rico. The disease was studied by determining the incidence of Alternaria spp. at the different stages of growth associated with foliage damage. Every 14 days, leaf samples of ‘Mercedes’ and ‘Excalibur’ were taken in experimental and commercial fields. A systematic experimental design in a zigzag pattern was used. Leaves segments were cut, disinfected, transferred to PDA with streptomycin and incubated at 27°C. After 7 days, the presence of Alternaria was determined. An incidence of 52% and 48% was obtained in 60- to 100-day-old plants of ‘Mercedes’ and ‘Excalibur’, respectively, in commercial fields. The incidence in experimental fields was lower, up to 33% and 25% on ‘Mercedes’ and ‘Excalibur’, respectively. Initial lesions were observed in 60–70 day old plants. A total of 280isolates were obtained. Of these, 32 selected ones were identified as A. destruens, A. tenuissima, A. palandui, and A. porn (A. alli).About 59% of selected isolates were placed within “semi-arborescent”, a group not described taxonomically.

The presence of Xylella fastidiosa was studied in 11 species of cicadellids of the cicadelleni tribe by DAS/ELISA test, using specific antibodies (Agdia Inc. Elkhart, IN). Collections were made in 2003–04 at nine coffee production zones of Costa Rica using vacuum directly over the crop. Specimens were counted, identified by species. They were placed in groups of two in 1.5-ml microfuge tubes and ground with a sterile toothpick in 200 µl of extraction buffer. All species contained bacteria in the foregut but only the species Kapateira nsp., Graphocephala bivittata, G. permagna, Fusigonalia lativittata, and Isogonalia adunca carried Xylella fastidiosa in more than 35% of the tested insects. Smaller samples of Dilobopterus instratus, D. hyalinatulus, Erythrogonia lecta, E. sonora, and Hortensia similis were analyzed and more need testing and confirmation by PCR.

A study was done in three coffee producing zones in 2002–2004. The zones were coffee monoculture (CM), coffee-citrus (CC), and coffee-avocado (CA). In each zone 4 plots of 40 plants were selected. In each plot 2 yellow sticky traps were used and changed weekly for 2 years. Trapped cicadellids were counted and identified. Similar population patterns of different species were seen in CA and CC but differences were found in the CM. Graphocephala bivittata (85%) and Erhytrogonia sonora (0.02%) were the most and less frequent species in the CM. Graphocephala permagna (34%) and E. sonora (0.11%) in the CC, and G. permagna (73.76%) and Macugonalia testudinaria (0.44%) in CA were found. In CC a greater variability and more equitable distribution of individuals (67.63%) was found. CM had the highest number of expected individuals. No similarity between the number of trapped individuals and the theoretical number in any zone was seen, which lead to an unbalanced population state.

Citrus leprosis, a damaging disease of citrus in South America, has recently moved northward into Central America and threatens citrus in Central and North America. Cytoplasmic and nuclear rhabdovirus-like particles have been associated with leprosis. Screening of a cDNA library, made from cytoplasmic ­leprosis infected tissue led to the identification of viral sequences. Northern hybridizations and RT-PCR assays showed an association only with cytoplasmic-leprosis. Northern blot analysis, using DIG-labeled DNA and RNA probes specific to Citrus leprosis virus sequences showed two banding patterns. Probes of clones from RNA1 hybridized with two RNAs of approximately 9–10 kb and 1.0–1.5 kb, while probes of RNA 2 clones hybridized with four RNAs of 4.7, 2.5, 1.6, and 1.0 kb. The genome-walking method for sequencing led to the entire sequence of RNA 1. RNA1 has two ORF’s. ORF1 codes for a large protein (276 kDa), with similarity to viral replicase polyprotein. The ORF2 codes for a small protein, with no similarity to known viral proteins.

The ‘Anaheim Disease’ of grapevines was described in the 1880’s and represented a crisis for California viticulture. As time passed and the industry adjusted the disease, now known as Pierce’s disease, became a sporadic nuisance rather than a crisis. Recently the disease has regained crisis status in California and the pathogen, Xylella fastidiosa, has emerged over the past decade from an obscure plant pathogen to one of the most feared ones. This followed the emergence of a serious disease of sweet orange in Brazil caused by a ‘new strain’ of the pathogen. This talk will discuss the history of the pathogen and how it emerged to threaten diverse horticultural industries. What factors have facilitated its spread, and what factors may facilitate its control? Will the available full-genome sequence lead to practical disease control? Or will the study of the ecology and environmental interactions of the pathogen provide avenues to control of the diseases caused by X. fastidiosa? These questions will be discussed and future research will be proposed with the goal of returning X. fastidiosa to a status of a sporadic nuisance rather than a crisis.

A method for the quantification of sweet orange and grapefruit stem pitting caused by Citrus tristeza virus.D. S. HOWD and R. H. Brlansky. University of Florida, Citrus Research and Education Center, 700 Experiment Station Rd., Lake Alfred, FL 33850.Citrus tristeza virus (CTV) isolates may cause a variety of host symptoms. One of the most severe is stem-pitting, which may reduce tree vigor, fruit yield, and size. Stem pitting is a consequence of the disruption of the cambium and abnormal growth of the phloem into the xylem. The determination of the amount of stem pitting usually involves a visual rating system where the results may vary. A more precise system is needed to determine the total area involved in pitting so as to correlate the effects of pitting on tree vigor and yield. In this study, we show the use of ASSESS image analysis software (L. Lamari, APS Press) for the quantification of percent area pitted in sweet orange and grapefruit affected by CTV isolates.

Analysis of the population diversity in selected Florida isolates of Citrus tristeza virus. A. S. Kahlon, K. L. Munjunath, and R. H. BRLANSKY. University of Florida, CREC, Lake Alfred, FL 33850.Citrus tristeza virus (CTV) affects citrus worldwide and symptoms vary from none to decline and death of trees on sour orange rootstock and/or stem pitting. Studies were done to molecularly characterize selected Florida CTV isolates to determine the effect of graft and aphid transmission (AT) on the population diversity. The diversity of three biologically different isolates was analyzed using multiple molecular markers (MMM) and the heteroduplex mobility assay (HMA). MMM is based on the RT-PCR amplification of genotype specific PCR products using primers sets derived from the analogous sites within the genomes of four different genotypic isolates. Amplified products give a profile for an isolate. Each of the three isolates studied was a mixture of isolates. A 403 bp region of the genome (nt 1082-1484) in the leader protease domain of the ORF 1 a was amplified and cloned using a pair of universal primers. HMA was done to detect genotypes using clones from the amplified PCR product of each isolate. Different CTV genotypes were present in each of the isolates, but only one was dominant. The isolates were graft and single AT and the populations were studied. The populations usually did not change upon graft transmission, but often changed after AT.

Citrus leprosis, a serious disease of citrus in South America, has recently spread into Central America. Serological detection is needed for disease management. Disease identification is based on visual symptoms and electron microscopy. We have demonstrated the presence of two distinct viruses, cytoplasmic and nuclear associated with the disease by nucleic acid hybridizations. With sequence information from the cytoplasmic type of Citrus leprosis virus (CiLV), a highly expressed gene was cloned into the bacterial expression vector, pET 27b. The leprosis protein with a carboxy-terminal fusion of HSV and 6x histidine tags was expressed in the expression host, E. coli, strain BL21. The expressed protein was purified using a Ni-NTA agarose column. Polyclonal antibodies were developed against the purified and insoluable proteins. A protein in cytoplasmic CiLV-infected plants but not in healthy plants was detected using Western blots.

Applications of Trichoderma formulations in horticulture. E. MONTE (1), I. Grondona (2), M. Gomez (2), A. Azpilicueta (2), A. Rodriguez (2), M. Rey (2), and A. Liobell (3). (1) Centro Hispano-Luso de Investigaciones Agrarias, University of Salamanca, Plaza Doctores de la Reina s/n, 37007 Salamanca, Spain; (2) NewBioTechnic, S.A. (NBT), Paseo de Bollullos de la Mitacion 6. Parque Industrial A-49 (PIBO) 41110 Bollullos de la Mitacion, Seville, Spain; (3) IBVF Isla de la Cartuja, CSIC/University of Seville, Spain.Trichoderma is an effective biocontrol for plant pathogenic fungi and viral vectors. Strain choice is important for effective and safe control. Trichoderma has many strategies for fungal antagonism and indirect effects on plants such as growth promotion, systemic resistance induction, and improved fertility. Some strains produce antibiotics and their suitability for control must be assessed. Other strains produce no antibiotics and may be useful in production systems since no adverse effects occur. We assessed natural strains and developed TUSAL®, a mixture of T. harzianum and T. viride, that effects pathogens. In the field it had effects on root development and production increased in trials. The impact of TUSAL® on beneficial organisms was assessed, and molecular methods developed to monitor strains and performance. Protein extracts were tested as biofungicides defining the concentration as fungicide effects. Protein production genes were introduced into organisms for lab production. Protein effect was studied separately, with conidia and with minimal fungicide doses. Both Trichoderma and proteins are patented and the formulation is being registered by NBT.

Methyl bromide (MeBr) soil fumigation is the main method for preplant disinfestation in strawberries. Pathogens and weeds are controlled and yields are good. MeBr environmental effects are unacceptable and alternatives are needed. IPM has been a IV FP EU-funded project to identify alternatives via biological, physical and low-chemical approaches to protection based on natural Trichoderma strainsactive against the fungal pathogens Collelotrichum, Phytophthora,and Botrytis,and to find strains of Trichoderma active with solarization. Combined approaches effectively control pests and diseases and minimize environmental damage. Other approaches are certification of plant material, solarization and chemicals, Trichoderma cell formulations, Trichoderma proteins, induction of systemic resistance, and soil-less systems.

Diseases caused by Xylella fastidiosa have been described as emerging problems and threats to grape production in North America and sweet orange production in Brazil. X. fastidiosa has been detected and isolated from grapes, coffee, and citrus in Costa Rica. Genetic analyses of local X. fastidiosa have been done using techniques such as RAPDs and repetitive element PCR. No Costa Rican isolates have been included and problems with reproducibility of RAPDs have been recognized. X. fastidiosa isolates from Costa Rica, Brazil, and the US were analyzed by variable number tandem repeats-PCR (VNTRs). Nine primer pairs for specific short sequence repeat (SSR) loci were used. Three primer pairs failed to render a product or amplification was inconsistent. Amplification of specific SSR loci was useful to distinguish strains from different geographic origins and hosts. Variability was found within North, Central and South American populations. The relationship of Costa Rican strains with those from South and North America will be determined.

Variability in colony morphology of Xylella fastidiosa isolates from Costa Rica and North America. M. MONTERO-ASTUA (1), J. S. Hartung (2), W. B. Li (2), E. Aguilar (1), C. Chacon (1), and C. Rivera (1,3). (1) Centro de Investigacion en Biología Celular y Molecular, Universidad de Costa Rica, San Pedro 2060; (2) USDA-ARS Fruit Laboratory, Beltsville, MD; (3) Facultad de Microbiologia.Xylella fastidiosa causes the diseases Pierce’s disease of grapes, citrus variegated chlorosis (CVC), and coffee leaf scorch (CLS). Different growth rates and colonies have been described. Isolates from different hosts in the US and Costa Rica had various morphologies on PW. Colony morphology was not associated with an isolate and colonies with two morphologies grew on the same plate. Subcultures of a specific morphology often produced colonies with different morphologies. Colonies showed similar growth rates, becoming visible 7–9 days. Single colonies tested with primers for X. fastidiosa (272-1-int/272-2-int) produced the expected product. Some were tested with universal primers (FD1/RP1) for bacterial 16S rDNA subunit and the products were digested with restriction enzymes (HinfI, TaqI, Sau3AI, HaeIII, AluI). The 16S rDNA products had the same digestion patterns. Cultures of X. fastidiosa can present variable colony morphologies. Conversion among morphologies was observed.

Three plasmodiophorid species, Polymyxa betae, P. graminis, and Spongospora subterranean are recognized vectors of economically important plant viruses. Some of these viral diseases were reported more than 50 years ago and latter their relationship to a plasmodiophorid vector was suggested. Some of these viruses are found worldwide and are now considered as crop threats. S. subterranean f. sp. subterranean, the cause of potato powdery scab and vector of Potato mop top virus, is special because both fungus and virus are considered as serious threats to potato production and are emerging diseases in the Americas. Chemical control of plasmodiophorids is erratic and resistance to certain viruses is under study. The molecular basis of transmission has been studied and viral genomes analyzed; however the virus-vector interaction, epidemiology and ecology are poorly understood. Development of molecular techniques for detection and quantification of these diseases promises to solve many questions.

Spongospora subterranean as a vector of Potato mop-top pomovirus in Costa Rica. M. MONTERO-ASTUA (1), V. Vasquez (1), W. Turechek (2), and C. Rivera (1,3). (1) Centro de Investigacion en Biologia Celular y Molecular, Universidad de Costa Rica, San Pedro 2060; (2) USDA-ARS Fruit Laboratory, Beltsville, MD 20705; (3) Facultad de Microbiologia.Spongospora subterranean f. sp. subterranean is the cause of potato powdery scab and vector of Potato mop-top pomovirus (PMTV). The vector and virus cause tuber blemishes and affect growth and yield. They are production threats in northern Europe and Australia and as emerging diseases in the Americas. S. subterranean and PMTV are soil-borne but their field association is not understood. In 2001–2004 a survey was done to assess occurrence and association in potato plantations. Paired tuber and leaf samples from 39 plantations were analyzed by ELISA using specific antibodies to S. subterranean and PMTV. The Jaccard index of association was 0.245 indicating a significant association (alpha = 0.05) according to a randomization test. PMTV soil transmission to bait plants was determined by bioassay, ELISA and PCR for PMTV and S. subterranean. Transmission to plants suggests soil contamination by PMTV bearing S. subterranean sporeballs or vector transmission. S. subterranean is an important factor of PMTV in Costa Rica.

Sugarcane yellow leaf was found in Costa Rica in 1994. Six years later, Sugarcane yellow leaf polerovirus (ScYLV) was confirmed in sugarcane by tissue printing, indirect ELISA, and immunosorbent electron microscopy. A geographical survey for ScYLV was done in 2000–2003 in 6 commercial plantation regions. Random sampling was done in 2000 one-hectare plots identified using GPS. Ten thousand 5–10 month old stalks were collected in plant and/or ratoon crops. DAS-ELISA was used to detect ScYLV. The average incidence of ScYLV was 43.0%. The Central Valley showed the major incidence (61.6%) while the South and Central Pacific regions had the lowest (27.6 and 27.8%, respectively). Incidences in Guanacaste, North- and Atlantic-region were 47.3%, 38.7%, and 43.8%, respectively. Incidence and distribution of ScYLV were developed using GPS.

Alternaria brown spot (ABS), caused by Alternaria alternate, produces necrotic spots on young leaves, fruits, and twigs, and corky lesions on fruit of tangerines and their hybrids. ABS was first reported in Australia in 1903 and subsequently in Florida in 1974, Israel in 1989, and South Africa and Cuba in 1992. New outbreaks have been reported in Turkey, Spain, Italy, Colombia, Brazil, Argentina, and Peru, and in Iran. Hybrids with Dancy mandarin as a parent, such as Orlando, Minneola, Fortune, Nova and Page, as well as Murcott tangor, are susceptible to ABS. Conidia of A. alternate are air-borne and their release is triggered by rainfall and/or sudden changes in humidity. The number of fungicide applications for disease control varies according to the variety and disease history. The Alter-rater model for timing fungicide sprays was developed in Florida and evaluated in Brazil. Ten or more fungicide applications per year may be necessary for good disease control and, thus, production of susceptible varieties may be limited to varieties that have sufficient value to justify control costs.

Trichoderma transcriptomics and its usefulness in plant protection. M. B. Rubio (1), M. R. Hermosa (1), R. E. Cardoza (1,2), S. Gutierrez (2), M. Rey (3), A. Liobell (4), and ENRIQUE MONTE (1). (1) Centro Hispano-Luso de lnvestigaciones Agrarias, University of Salamanca, Plaza Doctores de la Reina s/n, 37007 Salamanca, Spain, emv@usal.es; (2) Area of Microbiology, University of Leon, Ponferrada Campus, Ponferrada, Leon, Spain; (3) NewBioTechnic, S.A. (NBT), Paseo de Bollullos de la Mitacion 6. Parque Industrial A-49 (PIBO) 41110 Bollullos de la Mitacion, Seville, Spain; (4) IBVF Isla de la Cartuja, CSIC/University of Seville, Seville, Spain.Trichoderma is of biotechnological value, but its genome is poorly surveyed compared to other model microorganisms. Somestrains are used in industrial enzyme production, agriculture and bioremediation. It is less developed as a model to study and understand microbial interactions with plants and pests. Due to its ubiquity and rapid substrate colonization species have been used as biocontrol organisms for agriculture and their enzymes are used in industry. There is interest beyond its phenotype to exploit its genetic systems using functional genomics. TRICHOEST is a project, funded by the EU (FP5, QLRT-2001-02032), to analyze the transcriptome of selected strains with biocontrol potential, in regards to antagonism, nutrient stress and plant interactions. Selected clone sequences were done and cloned in appropriate vectors. Expression studies were performed. To determine the biological relevance of selected genes,strains lacking a functional copy of a given gene were obtained. Expression studies with DNA-arrays, containing PCR products from unigenes identified in T. harzianum 2413, were done using RNA probes from the wild type strain and transformants grown under simulated mycoparasitism.

Chayote (Sechium edule (Jacq.) Sw), a vegetable crop in Central America, is cultivated for local consumption and exportation. Since 2000, a new disease has been seen in plants in the Ujarras Valley, Cartago Province. The disease has affected 20% of the fields and caused 100% fruit loss because of induced fruit oxidation during post-harvest. Infected plants show white petioles and whiteness of fruits and stems, symptoms associated with phytoplasma diseases. Symptomatic plants were collected and tested by nested PCR and RLFP-PCR using universal primers, P1\P7 and R16R2\R16F2n, to amplify phytoplasma 16S rDNA sequences. Results suggest that a phytoplasma belonging to the 16SrI subgroup aster yellow is associated with the charyote whiteness disease which is different than the witches-broom disease of chayote reported previously. Partial sequence of the16S rDNA was deposited in GenBank (AY928382).

During 2004 a preliminary survey of 14 viruses was conducted in blackberry (Rubus spp.) plantations in Los Santos, Costa Rica. Only three viruses Tomato ringspot virus (ToRSV), Raspberry bushy dwarf virus (RBDV), and Tobacco streak virus (TSV) were detected by DAS-ELISA using specific antibodies and the procedures of Agdia Inc., Elkhart, Indiana. Afterwards, a symptomatic sampling in 15 plantations was done. A total of 236 samples were analyzed by DAS-ELISA against TSV, ToRSV, and RBDV. The majority of the plants (63%) were infected with RBDV. TSV and ToRSV were found in the 42% and 23% of the plants, respectively. The 37% of the plants presented single infection and the 43% presented mixed infections of two or the three viruses. Most common combination was RBDV and TSV. No plantation was virus-free; all had at least one of the three analyzed viruses.

In early 1990s a new disease in coffee in Desamparados and Los Santos (South region of San José) was found. Growers called it “crespera disease,” since foliar symptoms show malformation with curly narrows. The symptom was associated with zinc deficiency. Later Xylella fastidiosa was detected in symptomatic plants by DAS-ELISA and electron microscopy. The bacteria was cultured and detected by PCR. A survey was done in 2000 to determine the geographical distribution of the disease. 980 samples were collected and analyzed by DAS-ELISA. The bacteria were detected in most plantations. Simultaneously, the incidence of X. fastidiosa was evaluated in the fields. A survey was carried out to find coffee fields with the lowest incidence in the central region. In a plantation with 6% incidence four plots of 40 plants each were selected. Each plant in the plots was mapped and evaluated by ELISA every six months. After three years the incidence at the plots reach 100%, but only a few plants showed characteristic symptoms.